Living cells are principally comprised of proteins, carbohydrates and lipids. The plasma membrane enclosing cells and in fact all biological membranes are assemblies of lipid and protein molecules held together by noncovalent interactions. The three major types of lipids in cell membranes are phospholipids (the most abundant), cholesterol and glycolipids. All three are amphipathic, that is they have a hydrophilic ("water-loving" or polar) end and a hydrophobic ("water-hating" or non-polar) end.
When amphipathic molecules are surrounded on all sides by an aqueous environment, they tend to aggregate so as to bury their hydrophobic tails and leave their hydrophilic heads exposed to water. In so doing they can form any of the following structures: 1.) spherical micelies, with the tails inward, 2.) biomolecular sheets, or 3.) bilayers, with the hydrophobic tails sandwiched between the hydrophilic head group.
Most phospholipds and glycolipids spontaneously form bilayers in aqueous environments. Therefore, the formation of the lipid part of biological membranes is a self-assembly process. Moreover, such lipid bilayers tend to close on themselves to formed sealed compartments. For the same reason that lipid bilayers self-assemble, they self-seal when torn.
Although the lipid bilayer comprising a cell's plasma membrane is fluid, it is relatively impermeable and, therefore, serves as an effective barrier to the entry into the cytoplasm of a cell. This protective function is served by other biological membranes including mucous membranes (e.g. gastric mucosa and, nasal mucosa) and even skin.
However, for therapeutic purposes, it would be useful to get certain therapeutic agents through biological membranes. Liposomes are under investigation as vehicles for delivering therapeutic agents through biological membranes. Liposomes are spherical lipid structures that can be prepared in a manner that encapsulates water soluble biologically active molecules in the aqueous interior. When administered in vivo, liposomes fuse with biological membranes and thereby deliver the biologically active molecule contained within.
However, it has been found that liposomes injected into the body do not always deliver at the intended site and instead accumulate in the liver and spleen; and at sites of inflammation (Ostro, M. J. and P. R. Cullis, Am, J. Hosp. Pharm 46; 1576-1587 (1989)). Functionalized liposomes are being actively investigated as vehicles for targeted drug delivery. Galactosylated phospholipids, for example, have been incorporated in liposomes and used to deliver the liposomes specifically to asialoglycoprotein receptors of the hepatic system (Haensler, J. and F. Schuber, Glycoconjugate J. 1991,8, pp116-124). Immunoliposomes, constructed by covalent conjugation of antibodies to the phospholipid moieties on the liposomal surface, have also shown promise in targeting liposomes to specific cell tissues (Nassander, U. K., P. A. Steerenber, H. Poppe, G. Storm, L. G. PoeIs, W. H. De Jong, D. J. A. Crommelin, Canc. Res. 1992, 52, pp646-653, and Pinnaduwage, P. and L. Huang, Biochemistry 1992, 31, pp2850-2855).
In addition to targeting, the size of the liposomes have been reduced to improve their targeting and transfer through biological membranes. Straight chain lipid molecules have shown to be effective for delivering small peptides. For example, the phospholipid phosphatidyl ethanolamine conjugated to muramyl tripeptide to form (MTP-PE) has been found to be active in vivo and was found present in various organs 24 hours after injection, whereas the parent peptide was found to be excreted out of the body 60 minutes after injection (Fogler, W. E., R. Wade, D. E. Brundish, I. J. Fidler, J. Immunol. 1985, 135, pp1372-1377, and Phillips, N. C., J. Rioux, M. -S.Tsao, Hepatology 1988, 8, pp 1046-1050). These results suggest that the absorption of a peptide is enhanced by conjugation to a lipophilic moiety. Other agents that have been coupled to phospholipids include acyclovir (Welch, C. J., A. Lamson, A. C. Ericson, B. Oberg, R. Datema, J. Chattopadhyaya, Acta Chem. Scand. 1985, B39, pp47-54), ganglioside G.sub.M1 (Pacuszka, T., R. M. Bradley, P. H. Fishman, Biochemistry 30, pp2563-2570), oligosaccharides (Childs, R. A., K. Ddckamer, T. Kawasaki, S. Thiel, T. Mizuochi, T. Feizi, Blochem. J. 1989, 262, pp131-138), serum transferrin (Azelius, P., E. J. F. Demant, G. H. Hansen, P. B. Jensen, Biochim. Biophys. Acta 1989, 979, pp231-238), biotin, and fluorescent reagents.
Various methods have been described for derivatizing phospholipids to facilitate their conjugation with other molecules or moleties (for review, see Heath, T. D. and F. J. Martin, Chemistry and Physics of Lipids 1986, 40, pp347-358). However, each of these methods suffers from various difficulties in practical application. For example, one method comprises glutaraldehyde activation of phosphatidylethanolamine and ultimate conjugation to amines by reductive amination. The problem of dimerization both between the phospholipids and between proteins has made this method less than ideal. An alternative method comprises amide formation between phosphatidylethanolamine and the carboxyl terminus of a peptide or protein. However, this method suffers from low yields and formation of byproducts.
In yet another approach, the phospholipid and the protein are first activated and then reacted to form the conjugate. For example, Hutchinson et. al. describe a method in which a phosphatidylethanol-amine is activated with N-succinimidyI-S-acetyl-thioacetate (SATA) and treated with a hydroxylamine to yield a phospholipid-thiol derivative. The protein of interest is also activated with maleimide and then treated with the phospholipid derivative to form a stable conjugate via a thioether (Hutchinson et. al., FEBS Lett. 1986, 234, pp493-6). In a variation of this protocol, the phosphatidylethanolamine is activated with a maleimido moiety and the lysine residue of a protein is activated with a protected thiol (Loughrey, H. C. et. al., J. Immun. Methods 1990, 132, pp25-35). In practice, protocols employing these approaches are cumbersome to perform and the cost of the derivatizing agent is prohibitively expensive for scales above multigram quantities.
Phospholipid conjugates have also been formed by functionalizing phosphatidylethanolamine using a crosslinking reagent (e.g. dithiobis(succinimidyl propionate)) and reacting this intermediate with a lysine-containing protein so that the succinimidyl moiety is displaced by the amino group of the lysine residue (Afzelius, P., Blochem. Biophys. Acta 1989, 979, pp231-8). However, crosslinking reagents are not economically feasible for producing phospholipid conjugates, particularly on a large scale. In yet another method, phosphatidylethanolamine may be coupled to glycosylated proteins via the protein carbohydrate chain. For example, glycans can be oxidized with sodium periodate to give reactive aldehydes which can then be coupled to phosphatidylethanolamine via reductive amination with sodium cyanoborohyddde (Heath, T. et. al., Biochim. Biophys. Acta 1980, 599:42). This method is limited in its application only to glycoproteins and is often associated with low yields and byproduct formation.
None of the heretofore described methods offer a simple, generally applicable, efficient and economical (i.e. practical) means for generating phospholipid or other lipid conjugates.